Identification of new OPA1 cleavage site reveals that short isoforms regulate mitochondrial fusion

OPA1, a large GTPase of the dynamin superfamily, mediates fusion of the mitochondrial inner membranes, regulates cristae morphology, and maintains respiratory chain function. Inner-membrane-anchored long forms of OPA1 (l-OPA1) are proteolytically processed by the OMA1 or YME1L proteases, acting at cleavage sites S1 and S2 respectively, to produce short forms (s-OPA1). In both mouse and human, half of the mRNA splice forms of Opa1 are constitutively processed to yield exclusively s-OPA1. However, the function of s-OPA1 in mitochondrial fusion has been debated, because in some stress conditions, s-OPA1 is dispensable for fusion. By constructing cells in which the Opa1 locus no longer produces transcripts with S2 cleavage sites, we generated a simplified system to identify the new YME1L-dependent site S3 that mediates constitutive and complete cleavage of OPA1. We show that mitochondrial morphology is highly sensitive to the ratio of l-OPA1 to s-OPA1, indicating that s-OPA1 regulates mitochondrial fusion.

Identification of genes whose mRNAs are subjected to alternative splicing by endonuclease EndoG action in human and murine CD4+ T lymphocytes

The aim of this work was to identify genes whose mRNAs were subjected to alternative splicing by apoptotic endonuclease EndoG in CD4+ T lymphocytes from healthy humans, mice, and rats. In order to induce EndoG, lymphocytes were transfected with an EndoG-containing plasmid, or a control pGFP plasmid, or were incubated with cisplatin. Efficiency of transfection, number of cells with DNA damages and the level of EndoG expression have been monitored. Total cell mRNA has been sequenced and the changes in proportion of splice variants of genes were analyzed. The changes in the proportion of 28 mRNA splice variants have been identified in human and murine lymphocytes in both transfected with EndoG gene or incubated with cisplatin. Thus, EndoG can be considered as a potent modulator of alternative splicing of mRNA of identified genes.

Adverse early life environment induces anxiety-like behavior and increases expression of FKBP5 mRNA splice variants in mouse brain

Adverse early life environment (AELE) predisposes adult offspring toward anxiety disorders. Anxiety disorders are associated with prenatal injuries in key regions of the brain including prefrontal cortex (PFC), hippocampus (HP), and hypothalamus (HT). Injuries in these brain regions result in an impaired hypothalamus-pituitary-adrenal axis (HPA axis) and stress response. An important regulator of the stress response is FK506-binding protein 5 (FKBP5). FKBP5 is a cochaperone of the glucocorticoid receptor (GR) and inhibits GR-mediated regulatory feed-back on the HPA axis in response to stress. Human studies have shown that polymorphisms of FKBP5 are associated with higher FKBP5 levels. Increased FKBP5 leads to GR resistance and impaired negative feedback, which is associated with anxiety disorders. FKBP5 and its mRNA splice variants in the aforementioned brain regions have not been reported. We hypothesized that AELE will increase expression of FKBP5 and its mRNA splice variants in PFC, HP, and HT as well as increase anxiety in adult mice. AELE increased expression of FKBP5 and its mRNA variants in PFC, HP and HT at postnatal day 21. Additionally, AELE caused anxiety and increased GR abundance in association with these changes in FKBP5 expression. We speculate that these changes in FKBP5 mRNA variants affect HPA axis function and contributes to subsequent anxiety-like behavior later in life in AELE mice.

High Precision Detection of Rare Splice Isoforms Using Multiplexed Primer Extension Sequencing

ABSTRACTTargeted RNA-sequencing aims to focus coverage on areas of interest that are inadequately sampled in standard RNA-sequencing experiments. Here we present a novel approach for targeted RNA-sequencing that uses complex pools of reverse transcription primers to enable sequencing enrichment at user-selected locations across the genome. We demonstrate this approach by targeting hundreds to thousands of pre-mRNA splice junctions, revealing high-precision detection of splice isoforms, including rare pre-mRNA splicing intermediates.